Estudio de la reactividad vascular en aortas de ratones deficientes en APO-E

64 p.La aterosclerosis es una afección crónica, causa de diversas enfermedades cardiovasculares. Su etiología multifactorial desencadena un proceso pro-inflamatorio que termina en el daño de los vasos sanguíneos. La disfunción endotelial, caracterizada por un desbalance en la producción de factores relajantes y contráctiles derivados del endotelio, se reconoce como un paso previo al desarrollo de aterosclerosis.Se investigó la reactividad vascular en ratones deficientes en ApoE (ApoE+/- como modelo de dislipidemia y disfunción endotelial) alimentados con dieta normal y se los comparó con ratones Wild type (C57B1/6). Para esto se extrajo la arteria aorta de los ratones y se trabajó con la técnica de los anillos aórticos, la cual fue optimizada antes de comenzar la investigación. La reactividad vascular se midió a través la relajación de los anillos aórticos con acetilcolina (0,01, 0,1; 1, 5 y 10 μM) previa contracción con fenilefrina 5μM. La viabilidad de las preparaciones fue corroborada antes de la contracción con fenilefrina, mediante la aplicación al sistema de 60 mM de KCl.La técnica fue exitosamente optimizada y permitió llevar a cabo el estudio. Los resultados mostraron que los anillos aórticos de los ratones deficientes en ApoE contrajeron más que los WT frente a KCl (p = 0,0383), siendo la tensión de precarga óptima a 1,5 g para ambos grupos. La contracción con fenilefrina no mostró diferencias significativas entre ambos grupos, mientras que la relajación inducida por acetilcolina fue menor en ratones ApoE+/- que en ratones Wild type (p= 0,005). Estos datos sugieren una posible disfunción endotelial en los ratones ApoE+/-

Original Article Vascular hypercontractility and endothelial dysfunction before development of atherosclerosis in moderate dyslipidemia: role for nitric oxide and interleukin-6

Abstract: Atherosclerosis is a chronic disease that affects peripheral arteries and the aorta. Several inflammatory processes are required until the production of an atheroma. Before the atheroma appears, endothelial dysfunction is a key event. We hypothesized that endothelial dysfunction occurs in a mouse model of mild dyslipidemia, the mouse deficient in apolipoprotein E (apoE +/-). Using aortic rings preparation, we found that apoE +/-mice showed increased developed tension in response to KCl 60 mM when using a range a pre-loads from 0.5 to 2.0 grams (p = 0.038). Next, we tested the vasorelaxant capacity of apoE +/-aortas (pre-contracted with phenylephrine) in response to acetylcholine, an endothelium-dependent vasodilator. ApoE +/-aortas showed diminished vasorelaxation in a range of Ach concentrations (p = 0.0032). Next we assessed the levels of plasma NO metabolites, nitrite plus nitrate. These were significantly reduced, along with a significant decrease of the endothelial nitric oxide synthase in ApoE +/-mice. When we analyzed the morphology of the aortas in apoE +/-mice, these showed no signs of atheroma. In addition, we analyzed the levels of inflammatory cytokines, TNF-alpha, MCP-1 and interleukin 6 (Il-6). While TNFalpha was similar in both groups, (18.3 ± 2 pg/mL in wild type vs. 17.5 ± 2 pg/mL in apoE +/-), MCP-1 was increased in ApoE deficient mice (71.5 ± 0.8 pg/mL in wild type vs. 85.1 ± 7.4 pg/mL in ApoE +/-mice, p = 0.006), along with IL-6 (24.7 ± 1.7 pg/ml in wild type vs. 47.1 ± 12.5 in ApoE mice, p = 0.0055). These results suggest that mild dyslipidemia produces a pro-inflammatory state, associated with diminished NOS and NO production, which produces endothelial dysfunction

Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein <i>via </i>the MVB/lysosomal pathway

© FASEB. Brain regions affected by Alzheimer disease (AD) displaywell-recognized early neuropathologic features in the endolysosomal and autophagy systems of neurons, including enlargement of endosomal compartments, progressive accumulation of autophagic vacuoles, and lysosomal dysfunction.Although the primary causes of these disturbances are still under investigation, a growing body of evidence suggests that the amyloid precursor protein (APP) intracellular C-terminal fragment b (C99), generated by cleavage of APP by b-site APP cleaving enzyme 1 (BACE-1), is the primary cause of the endosome enlargement inADand the earliest initiator of synaptic plasticity and long-termmemory impairment. The aimof the present study was to evaluate the possible relationship between the endolysosomal degradation pathway and autophagy on the proteolytic processing and turnover of C99. We found that pharmacologic treatments that either inhibit autophagosomeformationorblock the fusionof autophagosomes to

Differences in social perception in people with schizophrenia and bipolar disorder

People with schizophrenia have difficulties recognizing other people's expressions, emotional states, and intentions; however, much less is known about their ability to perceive and understand social interactions. We used scenes depicting social situations to compare responses from 90 volunteers (healthy controls [HC], schizophrenia [SZ], and bipolar disorder [BD] outpatients from the Hospital del Salvador in Valparaíso, Chile) to the question: “What do you think is happening in the scene?” Independent blind raters assigned a score of 0 (absent), 1 (partial), or 2 (present) for each item based on whether the description identifies a) the context, b) the people, and c) the interaction depicted in the scenes. Regarding the context of the scenes, the SZ and BD groups scored significantly lower than the HC group, with no significant difference between the SZ and BD groups. Regarding the identification of the people and the interactions, the SZ group scored lower than the HC and BD groups, with no significant difference between the HC and BD groups. An ANCOVA was used to examine the relationship between diagnosis, cognitive performance, and the results of the social perception test. The diagnosis had an effect on context (p = .001) and people (p = .0001) but not on interactions (p = .08). Cognitive performance had a significant effect on interactions (p = .008) but not on context (p = .88) or people (p = .62). Our main result is that people with schizophrenia may have significant difficulties perceiving and understanding social encounters between other people

Proposed processing and turnover routes of C99.

<p>(A) (<i>i</i>) A small fraction of newly-synthesized APP in the endoplasmic reticulum (ER) can be a substrate of BACE1 that generates C99. Ubiquitinated (Ub) C99 can be a substrate of the endoplasmic reticulum-associated protein degradation (ERAD) pathway to ultimately be degraded by the proteasome. (<i>ii</i>) En route through the secretory pathway, a fraction of APP at the Golgi apparatus can also be a substrate of BACE1 that generates C99, which subsequently can be a substrate of γ-secretase (γ-sec) activity that generates Aβ peptides and cytosolic AICDγ, a proteolytic processing that can be inhibited by DAPT. (<i>iii</i>) Finally, within endo/lysosomal compartments APP can be degraded by acid hydrolases. (B) (<i>i</i>) Upon MG132 inhibition, ubiquitinated C99 accumulates within the ER. Ubiquitinated C99 can exit the ER and reach the Golgi apparatus. (<i>ii</i>) Both ubiquitinated C99 and C99 generated from APP can be cleaved at the Golgi apparatus by γ-secretase activity. Upon Brefeldin A (BFA) treatment, C99 can be relocated from the Golgi apparatus to the ER where it can be also cleaved by γ-secretase activity. (<i>iii</i>) Both APP and the excess of C99 can be degraded by acid hydrolases. (C) (<i>i</i>) Upon MG132 treatment, and (<i>ii</i>) the generation of an excess of C99 at the Golgi apparatus, (<i>iii</i>) chloroquine (CQ) treatment results in accumulation of both APP and C99 within endo/lysosomal compartments. For simplicity, other APP metabolites, such as sAPPβ, which is the other product of BACE1 activity on APP, or the C31 fragment, are not depicted.</p

C99 is degraded after redistribution to the endoplasmic reticulum.

<p>H4 cells stably expressing GFP-tagged C99-F/P-D/A were treated as follows: (A) with increasing concentrations of MG132 for 4 h; (B) left untreated or treated either with 1 µM DAPT for 16 h, 1 µM MG132 for 4 h, or 1 µM DAPT for 12 h followed by a combination of 1 µM DAPT and 1 µM MG132 for 4 h; (C) left untreated or treated for 4 h either with 5 µg/ml BFA, 1 µM MG132, or a combination of 1 µM MG132 and 5 µg/ml BFA; or (D) pretreated with 5 µg/ml BFA without or with 1 µM MG132 for 4 h followed by CHX-chase for 0–60 min without or with 1 µM MG132. (E) H4 cells stably expressing GFP-tagged APP-F/P-D/A were left untreated or treated for 4 h either with 5 µg/ml BFA, or a combination of 5 µg/ml BFA and 1 µM MG132. Cellular extracts were analyzed by immunoblot with anti-GFP antibody (A–E), or WO2 monoclonal antibody to detect C99 in cells expressing GFP-tagged APP-F/P-D/A (E). Immunoblot with anti-β-actin antibody was used as loading control. The positions of molecular mass markers are indicated on the left. (F) Densitometric quantification of the levels of C99 shown in E. Bars represent the mean ± SD (n = 4). *<i>P</i><0.05.</p

Different response of APP and C99 to CQ.

<p>(A–B) H4 cells stably expressing GFP-tagged APP-F/P-D/A (A) or C99-F/P-D/A (B) were left untreated or treated for 16 h either with 1 µM DAPT, 100 µM CQ, or with a combination of 1 µM DAPT and 100 µM CQ. Cellular extracts were analyzed by immunoblot with anti-GFP antibody. The positions of molecular mass markers are indicated on the left. (C) Densitometric quantification of the levels of APP and C99 shown in A and B. Bars represent the mean ± SD (APP n = 7; C99 n = 6). *<i>P</i><0.05. (D) Confocal fluorescence microscopy of H4 cells stably expressing GFP-tagged APP-F/P-D/A or C99-F/P-D/A left untreated (Control) or treated with 100 µM CQ for 16 h. Bar, 10 µm.</p

Intracellular localization and proteolytic processing of C99.

<p>(A) Schematic representation of GFP-tagged APP and C99 indicating their topological domains and the position of the HA tag, the Aβ peptide, the proteolytic cleavage sites (α, β and γ), and the AICDγ fragment. (B) Fluorescence microscopy analysis of H4 human neuroglioma cells transiently expressing APP-GFP or C99-GFP. Bar, 10 µm. (C–E) H4 cells transiently expressing C99-GFP were left untreated or treated for 16 h with 1 µM DAPT, labeled for 4 hr at 20°C with 1 mCi/ml [³⁵S]-methionine-cysteine, and chased at 37°C for the indicated times. C99 and Aβ species were immunoprecipitated from cell lysates with anti-GFP antibody (C), or from the culture medium with 6E10 antibody (E), respectively. Proteins were analyzed on 10%–20% Tricine gels and fluorography. The positions of molecular mass markers are indicated on the left. (D) Densitometric quantification of the levels of C99, C83, and AICDγ shown in C.</p

C99 is proteolytically cleaved in different sites.

<p>(A) Schematic representation of GFP-tagged C99, C83 and C31 indicating their topological domains, and the position of the Aβ peptide, the p3 peptide, the proteolytic cleavage sites (α, γ and caspase), and the AICDγ fragment. (B) H4 cells transiently expressing wild-type C99-GFP (WT) or C99-GFP with either the D87A mutation, the F38P mutation, or both (F/P-D/A), were left untreated or treated with 1 µM DAPT for 16 h. Cellular extracts were analyzed by immunoblot with anti-GFP antibody. The positions of molecular mass markers are indicated on the left.</p